The Arf6 GTPase regulates membrane traffic between the plasma membrane (PM) and an endosomal compartment and influences the dynamics of the cortical actin cytoskeleton. In many cells this pathway is the route followed by PM proteins that lack clathrin localization sequences and hence are endocytosed into cells independently of clathrin. We have demonstrated that PM proteins that are endocytosed via this Arf6-regulated, clathrin-independent pathway can either be recycled or routed to convergence with the "classical" early endosomal pathway and on to degradation in lysosomes. We followed the trafficking and fate of non-clathrin cargo molecules and compared it to PM proteins internalized by clathrin-mediated endocytosis. Among the PM proteins that enter cells through this non-clathrin pathway is major histocompatibility complex class I (MHCI). Non-clathrin cargo molecules like MHCI are initially observed in Arf6 and phosphatidylinositol 4,5-bisphosphate (PIP2)-associated endosomes that are distinct from the endosomes that contain clathrin cargo. Between 5 and 10 minutes, a fraction of the early endosomes containing non-clathrin cargo acquires phosphatidylinositol 3-phosphate (PI3P) and the early endosomal autoantigen 1 (EEA1) allowing it to fuse with the classical, Rab5, EEA1-associated early endosome. Another fraction of the Arf6 early endosome is recycled back to the PM. . The tubular endosomal membranes that recycle MHCI back to the PM have associated with them the Eps15 homology domain-containing protein, EHD1, which enhances MHCI recycling when overexpressed. Expression of constitutively active Arf6 mutant, Arf6Q67L, sequesters non-clathrin cargo in vacuoles that are enriched in PIP2 and blocks their trafficking to Rab5/EEA1 endosomes and degradation but does not affect trafficking of clathrin-derived cargo. These observations suggest that inactivation of Arf6, PIP2 turnover and acquisition of PI3P are required for fusion of endosomes arising from the Arf6 pathway with the Rab5/EEA1 early endosome system. Assays are being developed to reconstitute in vitro these membrane trafficking events.